Mice
Female 4–6 week-old BALB/cJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed at the University of Washington in an Institutional Animal Care and Use Committee (IACUC)-approved animal facility. All mice were used under an approved IACUC protocol (4317-01 to SCM) and in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.
DNA vaccination by gene gun
The Py circumsporozoite protein (CSP) DNA vaccine plasmids were constructed in the pUb.3 vector and co-administered with Escherichia coli heat-labile toxin (LT)-encoding plasmid adjuvant as described [19, 36–38]. The PyCSP-minigene encodes the SYVPSAEQI epitope and the PyCSP plasmid encodes the full-length CSP protein without the major repeat region. Supplementary Fig. 1 details amino acid sequences and agarose gel restriction digest plasmid validation for all PyCSP vaccines. All plasmid stocks were Sanger sequenced (GeneWiz Inc.) before use. Gene gun DNA vaccine cartridges were constructed as previously described [20, 37]. Mice were vaccinated on a shaved abdomen using a PowderJect-style gene gun by priming using two cartridges per day on Days 0 and 2 (0.5 µg DNA per cartridge). This method of priming with PyCSP/LT-encoding plasmids via gene gun is referred to as ggCSP.
Cryopreserved irradiated spz vaccination
Cryopreserved Py wild type (WT) 17XNL (cryo-RAS) were radiation-attenuated (100 Gy by C0-60), purified, vialed, and produced by Sanaria Inc. (Rockville, MD) [11, 39]. The vials were shipped to Seattle and stored in vapor phase liquid nitrogen per manufacturer recommendations. Cryo-RAS were thawed in a 37°C water bath for 30 seconds, diluted in Schneider’s insect media (Gibco, Thermo Fisher Scientific), and administered within 30 minutes of thawing. Spz counts were confirmed on a hemocytometer within one hour of injection. Figure legends specify the dose, volume, route, and number of injections for each experiment.
Freshly-dissected spz production and challenge
Female Anopheles stephensi mosquitoes infected with wild-type P. yoelii 17XNL (Py WT) were reared at Seattle Children’s Research Institute (Seattle, WA). Fresh spz were obtained by salivary gland dissection 14–18 days post-infection followed by Accudenz gradient purification as described [40]. Heat-killed spz (HK-spz) were generated by incubating Py WT spz in a 55°C water bath for 30 minutes. All spz were diluted in Schneider’s insect media for administration. Figure legends specify the dose, volume, route, and number of injections for each experiment. For all spz challenge administrations, 1x103 freshly dissected Py WT spz in 100 µL were injected retro-orbitally (RO) IV. Blood stage protection after spz challenge was assessed by Giemsa (Sigma-Aldrich) stained thin blood smear microcopy on Days 3–14 post-challenge. Mice were deemed protected if blood smears remained negative for parasites up to Day 14.
Intradermal and intravenous spz injections
ID injections in standard volumes (STV) of 10–50 µL were administered with a BD Veo Insulin Syringe with Ultra-Fine needle 6mm x 31G 3/10 mL/cc (#324909). STV injections were administered in two ID injections per dose on the lower back near the base of the tail. Ultra-low volume (ULV) ID injections of 2.5 µL were administered with a 10 µl Sub-microliter injection syringe (World Precision Instruments, Inc #NANOFIL) and a 36G Beveled needle (World Precision Instruments, Inc #NF36BV). ULV injections were administered in two ID injections per dose on the left rear footpad. IV injections were all administered RO in 100 µL with an Exel International Insulin Syringes with a 29G permanently attached needle. Supplementary Fig. 2 diagrams the locations of all ID and IV injections.
Glycolipid adjuvant preparation
7DW8-5 powder previously made under Good Manufacturing Practice (GMP) conditions was reconstituted in DMSO and prepared for injection as described [20]. 7DW8-5 or DMSO vehicle control was mixed with the cryo-RAS vaccines immediately before administration. All mice received 2 µg of 7DW8-5 adjuvant per immunization.
ELISA: Interferon-γ (IFN-γ) or IL-4 cytokine levels were determined by commercial ELISA kit according to manufacturer’s instructions (BioLegend, San Diego, CA; #430801 and #431104). Blood was collected into tubes containing EDTA and then plasma was isolated and frozen. For liver tissue, half of the liver was excised, weighed, and pulverized by bead beating in 3 mL lysis buffer (phosphate-buffered saline (PBS), 1:100 Pierce protease inhibitor (Thermo Fisher Scientific, # A32953), 0.05% Triton X-100). Homogenized samples were centrifuged at 16,000 x g for 10 min at 4°C. Supernatant was collected and frozen. All samples were diluted in the kit assay diluent, and absorbance was read on the CLARIOstar Plus plate reader (BMG Labtech, Germany) according to kit instructions. Standard curves and cytokine concentrations were calculated in Microsoft Excel.
PyCSP binding antibodies in mouse serum were determined by direct ELISA as previously described [41]. Blood was collected via submental bleed; serum was isolated and frozen. All serum samples were heat inactivated for 30 min at 56°C and centrifuged at 17,000 x g for 10 minutes prior to ELISA analysis. 50 ng per well recombinant PyCSP was plated in in 0.1M NaHCO3, pH 9.5, and incubated overnight at room temperature. Serum was diluted over a range of 1:50 to 1:109,350, and binding was detected with goat anti-mouse IgG Fc-HRP (Southern Biotech, #1013-05). Absorbance at 450 nm was determined with the BioTek ELx800 reader.
Depletion/Blocking antibodies
For CD1d and CD8 depletion/blocking studies, mice were injected intraperitoneally (IP) with 100 µg of anti-mouse CD1d (BioXcell, Lebanon, NH; #BE0000) or 500 µg of anti-mouse CD8 (BioXcell, #BE0061) 24 hours before challenge. Matched isotype controls were used at the same concentration respectively (BioXcell, #BE0088 (CD1d) or #BE0090 (CD8)). Additional animals were used to validate the depletion doses and schedule used for these studies (Supplementary Fig. 7). The depletion dose for CD8 was validated by whole blood leukocyte flow cytometry and CD1d dose was validated by plasma IFN-γ ELISA, as described below.
For CD8 depletion confirmation by flow cytometry, blood was collected via submental bleed into tubes containing EDTA 24 hours post CD8 depletion antibody or isotype injection. Whole blood was then resuspended in ammonium-chloride-potassium lysis buffer for 2–3 min to lyse red cells. The reaction was quenched with MACS buffer (PBS, 1 mM EDTA, 0.5% fetal bovine serum (FBS)). The final cell pellet containing whole blood leukocytes was resuspended in MACS buffer, blocked, stained, and fixed for flow cytometry as described below. The following Abs were used to assess CD8 cell depletion validation: live/dead dye-NIR, CD3e-BUV395, B220-BV711, CD4–Alexa Fluor, CD8a-BV421. Detailed information on flow reagents in Supplementary Table 1. Cell count per 100 µL blood was calculated based on known starting volume of mouse blood to normalize data. Flow cytometry was conducted on the LSRII instrument (BD Biosciences), and data were analyzed with FlowJo version 10.7.1 (BD Biosciences). For CD1d blocking confirmation, IFN-γ induced by 7DW8-5 was measured by ELISA. At 24 hours post CD1d or isotype depletion, 7DW8-5 was injected by the IV route. Six hours later, blood was collected (as described above), plasma was isolated, and IFN-γ cytokine levels were analyzed by ELISA was described above.
RAM2 spz-invasion blocking antibodies: RAM2 monoclonal antibodies were kindly provided by Noah Sather at Seattle Children’s Research Institute. RAM2 antibodies were produced and purified as described [41]. For spz-invasion studies, mice were injected IP with 150 µg of RAM2 or matched isotype control 24 hours before RAS immunization. Two hours post immunization, blood was collected via submental bleed and serum was isolated to quantify the amount of antibody circulating via ELISA, using RAM2 as a standard curve as previously described [42]. Serum was serially diluted over a range of 1:25 to 1:1,476,225 and binding was determined as described above with goat anti-mouse IgG-HRP (Southern Biotech, #1015-05). Standard curves for RAM2 were generated by nonlinear regression (log[agonist] vs response[three parameters]) in GraphPad Prism (San Diego, CA). Serum antibody concentrations were quantified by interpolating the average values from three different dilutions along the sample binding curve to the corresponding standard curves and multiplying by the dilution factor to determine the final concentration.
Parasite burden reverse transcription polymerase chain reaction (RT-PCR)
To quantify liver burden, half of the liver was excised, pulverized by bead beating into NucliSENS lysis buffer (bioMérieux), and nucleic acid was extracted as previously described [20, 43]. RNA was subjected to RT-PCR with the SensiFAST™ Probe Lo-ROX Kit (Bioline, London, UK) using a mouse GAPDH RT-PCR assay (IDT Inc, Coralville, IA) multiplexed with a Pan-Plasmodium 18S rRNA assay on a QuantStudio 5 real-time PCR machine (Thermo Fisher Scientific) as described [44]. Plasmodium 18S rRNA copy numbers per reaction were determined using a custom lot of quantified Armored RNA encoding full-length Plasmodium 18S rRNA (Asuragen, Austin, TX). To quantify popliteal draining lymph node (PO dLN) burden, the left PO dLNs were excised and pooled with alike PO dLN from the same group. Pooled PO dLNs were pulverized by bead beating in NucliSENS lysis buffer and processed for RT-PCR as described above.
Liver lymphocyte Isolation and flow cytometry
Liver lymphocytes were isolated by mechanical dissociation and Percoll density gradient as previously described [19, 45]. Briefly, livers were excised, mashed into a single cell suspension, and intrahepatic lymphocytes were isolated. Final liver lymphocyte pellets were transferred to a V-bottom 96-well plate for blocking, staining, and fixing for flow cytometry. All antibodies and staining conditions were as previously described [19, 20] and reagents are listed in Supplementary Table 1. Representative gating strategy is shown in Supplementary Fig. 6. Flow cytometry was conducted on the LSRII instrument (BD Biosciences), and data were analyzed with FlowJO version 10.7.1 (BD Biosciences).
Ex vivo IFN-γ ELISPOT
PyCSP peptide (SYVPSAEQI) was synthesized by Genemed Synthesis and reconstituted in DMSO. Mouse IFN-γ ELISPOT (eBioscience) was conducted by stimulating 5×105 splenocytes with CSP peptide (or DMSO vehicle control) at 1 µg/ml for 18 hr at 37°C and developed following manufacturer guidelines as reported previously [19, 46]. The number of spot-forming units (SFU) in each well was calculated using an ImmunoSpot 5.1 Analyzer (Cellular Technology Limited, OH). SFU were normalized to DMSO control wells and SFU per million splenocytes were reported.
nCounter® gene expression
Gene expression analysis was performed using the NanoString nCounter® Mouse Host Response Panel. Liver samples were prepared as described above for RT-PCR with n = 3 mice per group. Total RNA was extracted on the EasyMag system (bioMérieux) and the concentration was estimated with Nanodrop (Thermo Fisher Scientific). RNA (100 ng) was prepared for gene expression analysis at the Fred Hutchinson Cancer Research Center Genomics & Bioinformatics Core (Seattle, WA). Briefly, RNA samples were mixed with biotinylated capture and florescent reporter probes that were hybridized at 65°C for 12–16 hours. Hybridized samples were run on the NanoString nCounter® Mouse Host Response Panel using the recommended manufacturer protocol. After data collection, the nCounter® .RCC files were imported into nSolver Analysis Software 4.0 for review of quality control metrics, and the panel of housekeeping genes and positive controls was used to compute the normalization factor. Further data analysis was performed in RStudio version 2022.02.01 + 461 with R version 4.1.3. The normalized count matrix was evaluated for outliers using principal component analysis and no outliers were identified. Log2 transformed normalized counts per million were assessed for differential expression for ~ 0 + vaccine using limma version 3.50.3 [47]. Pairwise contrasts were performed for each vaccine group (IV-RAS, ID-RAS, ID-RAS + 7DW8-5) and control (ggCSP only). Significant genes were defined at FDR < 0.05 with BH correction and an absolute log2 fold change > 1 (Supplementary File 1). Selected pathways from MSigDB hallmark and KEGG collections [48, 49] were utilized to visualize differentially expressed genes.
Statistics
Comparisons of parasite burden RT-PCR, flow cytometry, and ELISPOT groups were done using non-parametric Kruskal-Wallis one-way analysis of variance with Dunn’s multiple comparisons test. ELISA data was analyzed with non-parametric Mann-Whitney test unless otherwise specified in the figure legend. Protection data was evaluated using Fisher’s exact test. All groups were compared against the ggCSP prime and 2x104 IV RAS trap positive control as a benchmark. Error bars in figures are reported as standard deviation (SD) of the mean with individual mouse samples shown if applicable. All p-values and individual experiment statistics are listed in corresponding figure legends. Statistical significance was defined as p < 0.05. Prism GraphPad Prism 9.1.2 Software (San Diego, CA) was used for all calculations, unless noted otherwise.